Covalent attachment of the hydrophilic polymer, poly(ethylene glycol), abbreviated “PEG,” to molecules and surfaces is of considerable utility in areas such as biotechnology and medicine. PEG is a polymer that possesses many beneficial properties. For instance, PEG is soluble in water and in many organic solvents, is non-toxic and non-immunogenic, and when attached to a surface, PEG provides a biocompatible, protective coating. Common applications or uses of PEG include (i) covalent attachment to proteins, e.g., for extending plasma half-life and reducing clearance through the kidney, (ii) attachment to surfaces such as in arterial replacements, blood contacting devices, and biosensors, (iii) as a soluble carrier for biopolymer synthesis, and (iv) as a reagent in the preparation of hydrogels.
In many if not all of the uses noted above, it is necessary to first activate the PEG by converting one or both of its hydroxyl termini, if it is a linear PEG, to a functional group capable of readily reacting with a functional group found within a desired target molecule or surface, such as a functional group found on the surface of a protein. For proteins, typical reactive amino acids include lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, tyrosine, the N-terminal amino group and the C-terminal carboxylic acid.
The PEG used as a starting material for most PEG activation reactions is typically an end-capped PEG. An end-capped PEG, in a linear PEG form, is one where one of the hydroxyl groups is converted into a non-reactive group, such as a methoxy, ethoxy, or benzyloxy group. In branched PEG structures having several hydroxyl end groups, one or more of them may be end-capped. Most commonly used is methoxyPEG, abbreviated as mPEG. End-capped PEGs such as mPEG are generally preferred, since such end-capped PEGs are typically more resistant to cross-linking and aggregation. The structures of two commonly employed end-capped PEG alcohols, mPEG and monobenzyl PEG (otherwise known as bPEG), are shown below,

wherein n typically ranges from about 10 to about 2,000.
Despite many successes, conjugation of a polymer to an active agent is often challenging. For example, attaching a relatively long poly(ethylene glycol) molecule to an active agent typically imparts greater water solubility than attaching a shorter poly(ethylene glycol) molecule. However, some conjugates bearing such long poly(ethylene glycol) moieties have been known to be substantially inactive in vivo. It has been hypothesized that these conjugates are inactive due to the length of the poly(ethylene glycol) chain, which effectively “wraps” itself around the entire active agent, thereby blocking access to potential ligands required for activity.
The problem associated with inactive conjugates bearing relatively large poly(ethylene glycol) moieties has been solved, in part, by using “branched” forms of a polymer derivative. Examples of a branched version of a poly(ethylene glycol) derivative are conventionally referred to as “mPEG2-N-hydroxysuccinimide” and “mPEG2-aldehyde” as shown below;

wherein n represents the number of repeating ethylene oxide monomer units. Other branched polymer structures comprise a polyol core, such as a glycerol oligomer, having multiple polymer arms covalently attached thereto at the sites of the hydroxyl groups. Exemplary branched polymer structures having a polyol core are described in U.S. Pat. No. 6,730,334.
Another reason for using branched structures like those above relates to the desire to increase circulation time of the polymer-bound drug. Larger polymers are known to have a longer circulation time than smaller polymers. Hence, drugs attached to the higher molecular weight polymers have longer circulation times, thus reducing the dosing requirements of the drug, which is often injected. There is also a practical aspect in the synthesis of the higher molecular weight polymeric reagents that favors the use of branched structures. As most mPEGs are synthesized by polymerization initiated by a small mPEG fragment, e.g. CH3CH2CH2O−Na+, any moisture present leads to the formation of PEG diol, a contaminant that leads to a difunctional PEG derivative. At a constant moisture content in the reactor, the amount of diol increases as the molecular weight of the final polymer is increased. Thus while the diol content is rather low with low molecular weight mPEGs, it is quite high with mPEGs with molecular weights around 30,000 Daltons or higher. Because branched PEGs are formed from smaller mPEGs, there is less difunctional influence on the higher molecular weight PEGs formed in this way than in comparable high molecular weight PEGs that are linear. Even in branched PEGs using mPEGs of medium molecular weights (˜20,000 Daltons) there are significant enough amounts of impurities introduced by diols to cause concern. Methods to remove these types of impurities typically involve chromatography of acidic or basic intermediates, e.g., see U.S. Pat. No. 5,932,462.
Multiarm polymers are especially attractive for the delivery of small drug molecules. By activating multiple arms at the termini, the overall polymer loading is increased. Thus, on a given multiarm polymer, the effective dose size per gram of polymer is doubled, tripled, quadrupled etc, as two, three, four, etc. arms, respectively, are conjugated with a small drug molecule. Despite the interest in using them for drug delivery, making these multiarm polymers truly useful has presented challenges. For example, the increased structural complexity of branching often results in a concomitant increase in synthetic complexity and/or purification difficulties. In the case of branched polymers based on polyol core molecules, the commercially available ethoxylated polyol cores are typically crude mixtures of oligomers of various molecular weights. For instance, pentaerythritol itself is available in high purity, yet commercially available ethoxylated derivatives are generally crude mixtures having highly variable chain lengths among the various arms. Invariably, there is one pentaeryritol arm that is largely unreacted in these mixtures and, because of the steric hindrance added by the three substituted arms, ultimate conversion of the fourth arm to a useable arm in a predictable manner is very difficult. Purification of these mixtures to give pure versions of a single multiarm, especially to eliminate the fraction having the unusable (unsubstituted) arm, is very difficult because common methods of purification, such as recrystallization, distillation, and chromatography, do not work for these highly viscous liquids or amorphous solids. The crude mixture of multiarm products that result from the use of the commercially available ethoxylated polyols are poorly suited for pharmaceutical applications where large polymer polydispersity values and structural variability are disfavored and high purity levels and a consistent composition must be achieved.
As a result, there is an ongoing need in the art for more readily synthesized and/or purified branched polymer derivatives that can be conveniently used in conjugation reactions with active agents. The present invention addresses this and other needs in the art.